Project summary/abstract: This work will build a contrast agent for the SARS-CoV-2 main protease (Mpro; also known as 3CLpro) and validate it in an animal model. This contrast agent will have significant value as a research tool because it will map and measure Mpro with spatial and temporal resolution. Conventional methods to studying SARS-CoV-2 are based on PCR and serology. These are powerful and affordable tools for population-wide studies but have limited value in research studies because they are in vitro tools that use single time-point sampling. They cannot monitor the biodistribution and time course of the viral load in vivo or detect the location of viral reservoirs. In contrast, in vivo imaging offers the ability to track biological phenomena longitudinally, quantitatively, and relatively non-invasively. Thus, Aim 1 of this proposal will build a contrast agent for Mpro based chemiluminescent resonant energy transfer (CRET). Chemiluminescence is a very useful in vivo imaging tool because it measures spontaneous emission of photons from contrast agents. Thus, the background emission of normal tissue is zero leading to high sensitivity imaging in contrast to in vivo fluorescence that suffers from high background. When in the CRET configuration, the probe is silent; activation via a Mpro-cleavable sequence leads to high signal. We will validate these CRET-based molecules with chemistry and recombinant Mpro. Aim 2 will validate this probe with a Sindbis virus models of SARS-CoV-2. We are using this Sindbis model because it is suitable for BSL-2 labs allowing work to proceed immediately unlike wild type SARS- CoV-2 requiring BSL-3. We will express Mpro via Sindbis virus in tissue culture and animal models and image viral progression in adult and neonate mice. After validating this contrast agent and imaging approach, the community will have a powerful tool to answer many important questions related to SARS-Cov-2 infection: What is the time course of infection and biodistribution?; How does biodistribution change by route of infection? Are there latent disease reservoirs?; How do protease levels change in response to therapy? This work is innovative because it will be the first example of in vivo Mpro imaging. The significance is motivated by the profound impact Covid-19 has had on our society. Importantly, the work is feasible based it will harness Dr. Jokerst?s extensive experience in chemistry and contrast agent development as well as Dr. Siqueira-Neto?s expertise in infectious disease including Zika virus.